The goal of this research is to increase our understanding of the ATP- dependent proteolytic systems in bacterial and animal cells. These pathways involve a new class of proteolytic enzymes which are very large, multimeric structures that cleave proteins and ATP in a linked reaction. A major goal will be to elucidate further the mechanisms of protease La (the lon product), which catalyzes the rapid degradation of many proteins in E coli. We hope to clarify the roles of ATP hydrolysis in allowing proteolysis, to learn how this enzyme preferentially recognized "abnormal proteins", and to localize functional domains on this model enzyme. In related studies we shall investigate why some heat-shock proteins with chaperonin functions (e.g. dnaK, the hsp70 homolog) are required for rapid degradation of certain mutant proteins by protease La. In eukaryotic cells, the degradation of many proteins involves conjugation to ubiquitin (Ub), which marks them for rapid degradation by 26S (1500kDa) ATP-requiring complex. We hope to understand the structural organization and functional properties of this complex. In muscle extracts, the 26S complex can be formed by the association of two smaller ATP-dependent protease complexes: the 700kDa proteasome, and a newly discovered 500kDa activity, multipain, which we plan to characterize in depth. By contrast, in ATP-depleted reticulocytes, the 26S complex dissociates reversibly into proteasomes and two factors, CF-1 and CF-2, which we found to activate and inhibit (respectively) proteasome function. Our major goals will be to understand how these components interact to allow degradation of Ub- proteins and to clarify the interrelationships between these different components of the 26S complex. We also hope to elucidate the roles of ATP in their function and to learn whether these enzymes utilize similar mechanisms as the bacterial ATP-hydrolyzing proteases.